This project focuses on a very challenging problem in the medical community, the development of treatments for drug- resistant bacterial infections. The leading cause of clinical complications in the United States is nocosomial infections caused by the drug-resistant pathogen, Staphylococcus aureus. There is currently a dire need for new drugs to be developed for controlling these infections. Towards this goal, our proposal discusses efforts to identify the biochemical and chemical basis for antibacterial activity of N-thiolated beta-lactams, a new class of antibiotics discovered in our laboratory at University of South Florida. Preliminary data on more than 50 active analogues indicates that these substances possess antimicrobial behavior selective for Staphylococcus bacteria, with enhanced activity towards multi-drug resistant strains (MRSA). Unlike all previously known beta-lactam drugs, these compounds appear to affect early developmental events during cell replication, not cell wall crosslinking. These compounds have highly unusual structure-activity profiles which need to be explored further. Electron microscopy experiments indicate that our lactams produce no morphological defects in MRSA cells, or cytotoxic effects in human fibroblasts. The compounds are stable over a wide pH range (pH 1 to 10), and are totally transparent to penicillinases as well as to most chemical reagents we have studied. Our Proposed Studies consist of three Specific Aims. Aim 1 is to study the biochemical and chemical basis for antimicrobial activity, and will include (1) electron microscopy experiments to look for morphological defects in bacterial cells due to damage by the lactams, and to identify where the drug accumulates in the cell, (2) radiolabeling to determine which of the primary cellular processes (cell wall synthesis, protein synthesis, or nucleic acid synthesis) are affected by the beta-lactams, and the means by which the drugs function chemically, and (3) studies to define whether the lactams bind covalently or non-covalently to the biological target, and to identify the cellular target. Aim 2 is to assess further whether the lactams are cytotoxic to mammalian cells. Aim 3 is to develop new approaches to solid phase synthesis of affinity resins (for experiments on isolating the cellular target) and lactam libraries (for expanded drug screening). We also aim to develop novel prodrug delivery systems for the prevention and treatment of MRSA infections. We believe that these studies will provide extraordinary opportunities to develop new therapeutics and approaches for the control of hospital-borne drug-resistant infections.